Influence of DOTA chelator position on biodistribution and targeting properties of ¹¹¹In-labeled synthetic anti-HER2 affibody molecules

Affibody molecules are a class of affinity proteins. Their small size (7 kDa) in combination with the high (subnanomolar) affinity for a number of cancer-associated molecular targets makes them suitable for molecular imaging. Earlier studies demonstrated that the selection of radionuclide and chelator may substantially influence the tumor-targeting properties of affibody molecules. Moreover, the placement of chelators for labeling of affibody molecules with ^99mTc at different positions in affibody molecules influenced both blood clearance rate and uptake in healthy tissues. This introduces an opportunity to improve the contrast of affibody-mediated imaging. In this comparative study, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to the synthetic affibody molecule Z_HER2:S1 at three different positions: DOTA-A1-Z_HER2:S1 (N-terminus), DOTA-K58-Z_HER2:S1 (C-terminus), and DOTA-K50-Z_HER2:S1 (middle of helix 3). The affinity for HER2 differed slightly among the variants and the K_D values were determined to be 133 pM, 107 pM and 94 pM for DOTA-A1-Z_HER2:S1, DOTA-K50-Z_HER2:S1, and DOTA-K58-Z _HER2:S1, respectively. Z_HER2:S1-K50-DOTA showed a slightly lower melting point (57 °C) compared to DOTA-A1-Z_HER2:S1 (64 °C) and DOTA-K58-Z_HER2:S1 (62 °C), but all variants showed good refolding properties after heat treatment. All conjugates were successfully labeled with ¹¹¹In resulting in a radiochemical yield of 99% with preserved binding capacity. In vitro specificity studies using SKOV-3 and LS174T cell lines showed that the binding of the radiolabeled compounds was HER2 receptor-mediated, which also was verified in vivo using BALB/C nu/nu mice with LS174T and Ramos lymphoma xenografts. The three conjugates all showed specific uptake in LS174T xenografts in nude mice, where DOTA-A1-Z_HER2:S1and DOTA-K58-Z_HER2:S1 showed the highest uptake. Overall, DOTA-K58-Z _HER2:S1 provided the highest tumor-to-blood ratio, which is important for a high-contrast imaging. In conclusion, the positioning of the DOTA chelator influences the cellular processing and the biodistribution pattern of radiolabeled affibody molecules, creating preconditions for imaging optimization.